In the last five years our laboratory has identified the genetic basis of two dominantly inherited autoinflammatory disorders. The TNF receptor-associated periodic syndrome (TRAPS) is characterized by oftentimes prolonged attacks of fever, serositis, migratory rash and myalgia, arthritis, periorbital edema, conjunctivitis, and, in some patients, systemic amyloidosis. Because it was first recognized in a large family of Scottish-Irish ancestry, this condition had initially been called ?familial Hibernian fever.? In 1999 our group identified the first six mutations in the gene encoding the 55 kDa tumor necrosis factor receptor (TNFRSF1A) in seven families of various ancestries with this clinical condition, including the original ?Hibernian? family, and we therefore proposed the more ethnically neutral TRAPS nomenclature currently in use. Five of the initial six mutations were missense substitutions at extracellular cysteine residues, resulting in the disruption of highly conserved disulfide bonds. In three members of a family with the C52F mutation, we found a defect in the activation-induced shedding of the p55 (but not the p75) TNF receptor, possibly leading to impaired homeostasis in the inflammatory response. Subsequently, our laboratory has identified additional TRAPS mutations, studied genotype-phenotype correlations, and established p55 receptor shedding defects for most, but not all, mutations tested. We also conducted an open-label, dose-escalation study that supported the efficacy of the TNFR p75:Fc fusion protein, etanercept, in TRAPS, and we developed TRAPS knockin mice for further mechanistic studies. In 2002 we and a French group independently discovered dominantly inherited de novo mutations in a second gene, CIAS1 (also known as NALP3 or PYPAF1), that cause a distinct disorder known as neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and articular (CINCA) syndrome. Manifestations of NOMID/CINCA may include daily fevers, an urticaria-like skin rash, chronic aseptic meningitis, uveitis, papilledema, sensorineural hearing loss, mental retardation, patellar and epiphyseal long bone overgrowth, and systemic amyloidosis. In our initial cohort of 13 patients, we identified mutations in 6, all in exon 3, which encodes the NACHT (or nucleotide binding site) domain of the cryopyrin protein. Consistent with role of cryopyrin in IL-1 regulation, we found evidence of increased monocyte IL-1beta by Western blot in a mutation-positive patient, compared with healthy controls. In subsequent studies we have identified additional CIAS1 mutations in NOMID/CINCA patients, but have not found mutations in several related proteins among CIAS1 mutation-negative patients. Results of the Last Year Mutational analyses of the TNFRSF1A gene: In a study of three families and one sporadic case of TRAPS, we identified two novel mutations (C52S and S74C) and one mutation recently described by another group (C30Y). This brings the total number of TRAPS-associated TNFRSF1A mutations to 36, 16 of which are missense substitutions at extracellular cysteine residues. C52S and C30Y are the second and third mutations identified at each of the respective residues, indicating that these sites are mutational ?hot spots.? Although cysteine mutations have been associated with more severe disease and a predilection to systemic amyloidosis, as has been seen in patients with both the C30R and C30Y mutation, we observed mild disease in several individuals with C30Y, thus suggesting variable penetrance. We also studied the frequency of two low-penetrance TNFRSF1A mutations in large cohorts. The R92Q variant is the most frequent mutation found in our NIH cohort of symptomatic TRAPS patients (approximately 33%), and has also recently been found at increased frequency in an early arthritis clinic and in French patients with atherosclerotic disease. In our present studies, we found this variant in 3.8% of 712 Caucasian controls, consistent with its possible role in more common genetic disorders. We also studied the population frequency of P46L, a second low-penetrance mutation we had previously identified. P46L was found in 0.3% of 738 Caucasian controls, but in 16% of 152 African-American samples. Studies of TNFRSF1A knockin mice: During the previous reporting period we developed two lines of mice harboring two different TRAPS-associated mutations, C33Y and T50M. On a mixed C57BL6/129 background, we confirmed the respective knockin mutations by DNA sequencing. RT-PCR from both T50M and C33Y mice demonstrated Tnfrsf1a transcripts of the appropriate size, and immunoprecipitation and Western blotting with anti-mouse Tnfrsf1a showed intact 55 kDa receptor in leukocytes from homozygous T50M and C33Y mice. Our previous studies of homozygous knockin mice demonstrate a phenotype similar to p55 receptor knockouts, suggesting a possible defect in trafficking of the mutant receptor to the cell surface. Studies of endotoxin-induced hypothermia conducted during the current reporting period do indicate a more pronounced inflammatory response in heterozygous T50M mice. Genetic studies in NOMID/CINCA: During the previous reporting period we screened eight patients with clinical NOMID/CINCA for CIAS1 mutations. Four were mutation-positive, including two with novel mutations (G326E and F523C). In the present reporting period we have screened an additional 13 patients, 9 of whom are positive for CIAS1 mutations. Among this new group, there are three novel missense changes in four patients: V262A, F443L, and R488K. Overall, among 34 patients with clinical NOMID/CINCA we have found mutations in only 19 (56%). With the new mutations identified in this study, the total number of known disease-associated CIAS1 variants is now 35. Among our 15 mutation-negative patients, we have screened a number of genes encoding proteins in the same family as cryopyrin, including NALP1 (DEFCAP), NALP2 (PYPAF2), NALP4 (PYPAF4), and NALP12 (PYPAF7), as well as the ASC adaptor protein, but have not identified any mutations. Therapeutic trial of anakinra in NOMID/CINCA: Dr. Raphaela Goldbach-Mansky, a collaborator in the Office of the Clinical Director, is currently conducting an open-label therapeutic trial of anakinra, the IL-1 receptor-antagonist, in NOMID/CINCA. The trial includes a baseline observation period, a treatment period initially with a dose of 1 mg/kg/day and escalating to 2 mg/kg/day for symptoms or laboratory findings, a brief washout, and an open-label extension. A total of 18 patients have been enrolled, 11 of whom have completed the washout. The response to anakinra was rapid and dramatic, including marked improvement in symptoms, rash, CSF opening pressure, and acute phase reactants (erythrocyte sedimentation rate, C-reactive protein, and serum amyloid A). With drug withdrawal, clinical symptoms returned and acute phase reactants increased. Conclusions and Significance Taken together, our data confirm a major role for abnormalities in cytokine signaling in the dominantly inherited periodic fevers. TNF and IL-1 are the two major pyrogenic cytokines in man, and we have now found pathogenic mutations in both signaling pathways. Our previous successful trial of etanercept in TRAPS, and the present experience with anakinra in NOMID, confirm the clinical importance of these observations. During the next year, our objectives will be: 1) to continue mutational studies of genes in the TNF and IL-1 pathways in patients with uncharacterized inflammatory disorders; 2) to continue physiologic studies of TRAPS knockin mice; 3) to develop NOMID/CINCA knockin mice; and 4) in collaboration with Dr. Goldbach-Mansky, to continue therapeutic trials of IL-1 inhibition in CIAS1 disease.